Sustained release hydrophobic bioactive PLGA microspheres

ABSTRACT

A controlled release microcapsulate pharmaceutical formulation for burst-free, sustained, programmable release of hydrophobic bioactive agent over a duration from 24 hours to 100 days comprising: and a blend of end-capped uncapped biocompatible, biodegradable poly(lactide/glycolide).

CROSS-REFERENCE

This application is a continuation application of U.S. patentapplication Ser. No. 08/920,326, filed Aug. 21, 1997 now U.S. Pat. No.6,447,796 which a continuation-in-part of U.S. patent application Ser.No. 08/698,896 filed Aug. 16, 1996 now U.S. Pat. No. 5,705,197, which inturn is a continuation-in-part of U.S. patent application Ser. No.08/242,960 filed May 16, 1994 now U.S. Pat. No. 5,693,343; U.S. patentapplication Ser. No. 08/675,895 filed Jul. 5, 1996 now U.S. Pat. No.6,217,911; and U.S. patent application Ser. No. 08/789,734 filed Jan.27, 1997 now U.S. Pat. No. 6,309,669 which in turn is acontinuation-in-part of U.S. patent application Ser. No. 08/590,973filed Jan. 24, 1996 now abandoned which in turn is acontinuation-in-part of U.S. patent application Ser. No. 08/446,148filed May 22, 1995 now abandoned and U.S. patent application Ser. No.08/446,148 filed May 22, 1995 now U.S. Pat. No. 6,410,056, andincorporates in its entirety, the contents of U.S. patent applicationSer. Nos. 08/698,896; 08/242,960; 08/675,895; 08/789/734; 08/590,973;08/446,149 and 08/446,148.

GOVERNMENT INTEREST

The invention described herein may be manufactured, licensed and used byor for governmental purposes without the payment of any royalties to usthereon.

FIELD OF THE INVENTION

This invention relates to providing novel pharmaceutical compositionsfor local delivery and burst-free programmable, sustained release ofhydrophobic drugs from biocompatible, biodegradable poly(DL-lactide-co-glycolide) (PLGA) microspheres. The resulting product isadministered locally into soft tissues by sub-cutaneous orintra-muscular injection where it will locally sustain release the drug.

This invention relates generally to providing novel blend of end-cappedand uncapped providing novel biocompatible and biodegradable PLGAmicrospheres for burst-free programmable sustained release ofhydrophobic biologically active agents which degrade over a period of upto 100 days in an aqueous physiological environment. The active agentscontemplated within the scope of this invention includes the delivery ofpoly-peptide antibiotics, antimalarials, antituberculosis drugs,anesthetics, analgesics, anticancer agents, antiparasitic agents,antibacterial agents, antifungal agents, antiinflammatory agents,immunosuppressive agents, immunostimulatory agents, dentinaldesensitizers, odor masking agents, nutritional agents, antioxidants,and insulins.

The invention also relates especially to providing novel pharmaceuticalcompositions for sustained release of compounds for treating cancer,inflammatory and/or autoimmune disorders from PLGA microspheres.

BACKGROUND OF THE INVENTION

This invention is particularly effective for the localized delivery ofchemotherapeutic hydrophobic anticancer agents, inclusive ofpaclitaxel(taxol)doxorubicin, 5-fluorouracil, campthothecin, cisplatin,and metronidazole, their corresponding derivatives and functionallyequivalents, and combinations thereof from PLGA microspheres.

One-third of all individuals in the United States of America (U.S.)alone will develop cancer.

Although the five-year survival rate has risen dramatically to nearlyfifty percent as a resulting progress in early diagnosis and thetherapy, cancer still remains second only to cardiac disease as a causeof death in the U.S. twenty percent of Americans die from cancer, halfdue to lung, breast, and colon-rectal cancer.

Breast cancer is the second leading cause of death in women in the U.S.Approximately 135,000 women are diagnosed with and 42,000 women die frombreast cancer annually (1). Breast cancer treatment plans include acombination of surgery, radiotherapy, and chemotherapy (CT). The generaltreatment plan for stage I and II breast cancer is conservative surgeryand radiotherapy (2,3,4,5). The general treatment plan for stage III andIV breast cancer is a combination of surgery, radiotherapy, and systemicCT using chemotherapeutic agents such as taxol (6,7,8,9,10).

Taxol treatment is recommended for the treatment of breast cancer whenCT for metastatic disease has failed or when disease relapse hasoccurred within 6 months of adjuvant CT. Advantages of taxol treatmentinclude: (1) lack of cardiotoxicity; (2) a mechanism of action, thestabilization of microtubules, which targets a large percentage of tumorcells, as opposed to normal cells; and (3) inhibition of angiogensis(11,12). Taxol is systemically administered intravenously (i.v.),primarily as a bolus administration. Systemic CT using taxol, as well asother chemotherapeutic drugs, is highly effective, in terms ofadditional years of life gained as a result of therapy; however, thereare many problems associated with this treatment regimen.

CT drugs are high by cytotoxic (13), and typically, large doses of CTdrugs are needed to produce an optimal therapeutic response(13, 14);therefore, CT drugs have a low therapeutic index(13). Side effectscommonly seen with taxol CT include: nausea, vomiting, fever, weightchanges, musculoskeletal pain, neuropathy, general malaise, immunedysfunction, and the development of tumor resistance to taxol (9,15,16).

An additional side effect seen in patients treated with taxol is asevere hypersensitivity reaction due to Cremophor EL, taxol'ssolubilizing agent (17). This side effect is controlled via patientpre-medication using a combination of corticosteroids, antihistamines,and histamine receptor antagonists. Often, patients become so ill duringtherapy that they are removed from treatment regimens or that drugdosages are lowered. The consequence of these regimen changes isfluctuating drug levels, which equates to decreased efficacy.

The problems associated with systemic taxol treatment signal the needfor the development of a drug delivery system which offers a safer and amore effective means of administering toxic agents, such as taxol, tobreast cancer patients, as well as to other cancer patients.

Delivery systems based on prolonged exposure to taxol have beeninvestigated as a means to overcome the problems associated with bolusadministration of taxol. These systems include infusional administrationof taxol over 1,3,24, or 96 hours and administration of taxol viapolymeric carrier vehicle. Infusional data suggests that cytotoxicitymay be enhanced due to the increased exposure of cycling cells and hasshown, in vitro, 4.4 fold less resistance in multi-drug resistant MCP-7human breast carcinoma cells exposed to taxol for 24 hours as comparedto 3 hours (16). A 96-hour taxol infusion study in patients withmetastatic breast cancer showed that this infusion schedule: (1) wasbetter tolerated than bolus administration of taxol, as evidenced bymild side effects, such as nausea and myalgia; (2) did not causesignificant hypersensitivity reactions despite the omission ofcorticosteroid pre-treatment; (3) did not result in any cardiac, renal,or hepatic toxicity, and (4) resulted in major objective responses in7/26 patients (26.9%), with a 6 month median response duration (16). Inthis trial, the predominant toxic side effect was granulocytopenia whichresulted in taxol dose reduction in 3/26 patients (11.54%) and inhospitalization of 4/26 patients (15.38%).

Taxol infusion regiments offer significant advantages over bolusadministration of taxol in terms of systemic toxicity, efficacy, andresistance; however, immune dysfunction still appears to be the majorlimiting factor in the success of this treatment regimen.

Certain chemotherapeutics such as paclitaxel (taxol) and camptothecin,which are efficacious when administered systemically must be deliveredat very high dosages in order to avoid toxicity due to poorbioavailability. For example, paclitaxel (taxol) has been usedsystemically with efficacy in treating several human tumors, includingovarian, breast, and non-small cell lung cancer. However, maintenance ofsufficient systemic levels of the drug for treatment of tumors has beenassociated with severe, in some cases “life-threatening” toxicity, asreported by Sarosy and Reed, J. Nat. Med. Assoc. 85(6):427-431 (1993).Paclitaxel is a high molecular weight (854), highly lipophilicdeterpenoid isolated from the western yew, Taxus brevifolia, which isinsoluble in water. It is normally administered intravenously bydilution into saline of the drug dissolved or suspended inpolyoxyethylated castor oil. This carrier has been reported to induce ananaphylactic reaction in a number of patients (Sarosy and Reed (1993) soalternative carriers have been proposed, such as a mixed micellarformulation for parenteral administration, described by Alkan-Onyuksel,et al., Pharm. Res. 11(2), 206-212 (1994). There is also extensivenon-renal clearance, with indications that the drug is removed andstored peripherally. Pharmacokinetic evidence from clinical trials(Rowinsky, E. K., et al., Cancer Res. 49:4640-4647 (1989) and animalstudies (Klecker, R. W., Proc. Am. Cancer Res. 6.43:381 (1993) indicatesthat paclitaxel penetrates the intact blood-brain barrier poorly, if atall, and that there is no increased survival from systemicintraperitoneal injections of paclitaxel into rats with intracranialgliomas. Paclitaxol has been administered in a polymeric matrix forinhibition of scar formation in the eye, as reported by Jampel, et al.,Opthalmic Surg. 22, 676-680 (1991), but has not been administeredlocally to inhibit tumor growth.

SUMMARY OF THE INVENTION

This invention provides a method and novel pharmaceutical compositionsfor local delivery and burst-free programmable sustained release ofhydrophobic drugs from biocompatible, biodegradble poly(DL-lactide-co-glycolide) (PLGA) microspheres. The hydrophobic drugs arereleased over a time period while at the same time preserving itsbioactivity and bioavailability.

It is therefore an object of the present invention to provide achemotherapeutic composition and method of use thereof which providesfor effective long term release of chemotherapeutic agents that are notstable or soluble in aqueous solutions or which have limitedbioavailability in vivo for treatment of solid tumors.

It is a further object of the present invention to provide a compositionand method of use for the treatment of solid tumors withchemotherapeutic agents that avoids high systemic levels of the agentand associated toxicities.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 shows the microsphere morphology of taxol PLGA: 10% core loadformulation; Background Count Specifics: 426 particles; mean=5.05 um;median=2.74 um Taxol/PLGA Microspheres Counted: 230,046; Mean=7.75 um;Median=6.15 um;

FIG. 2 shows the in vitro release kinetics of taxol/PLGA: 10% core loadformation; Summary of Taxol Release; (1) Amount of taxol released over10 days (based on supernatant data)=7.06% (749.13 ug). (2) Amount oftaxol released over 10 days (based on residual data); Residual Taxol at10 days=59.81% (6350.5 ug); 100%−59.81%=40.19% released over 10 days.(3) Time for complete PLGA degradation=approximately 20 days.

FIG. 3 shows the microsphere morphology of taxol/PLGA: 20% core loadformulation; Background Count Specifics; 103 particles; mean=3.39 um;median=1.78 um; Taxol/PLGA Microspheres Counted: 84,768; Mean=6.43 um;Median=4.69 um;

FIG. 4 shows the in vitro release kinetics of taxol/PLGA: 20% core loadformulation; Background Specifics; 111 particles; mean=8.15 um;median=3.58 um; Taxol/PLGA Microspheres Counted:132,846; Mean=6.00 um;Median=3.99 um;

FIG. 5 shows the microsphere morphology of taxol/PLGA: 40% core loadformulation; Background specifics: 171 particles; mean=8.15 um;median=2.46 um; Taxol/PLGA Microspheres Counted: 116,422; Mean=5.69 um;Median=3.78 um;

FIG. 6 shows the in vitro release kinetics of taxol/PLGA: 40% core loadformulation;

FIG. 7 shows the microsphere morphology of taxol/PLGA: 50% core loadformulation;

FIG. 8 shows the in vitro release kinetics of taxol/PLGA: 50% core loadformulation;

FIG. 9 shows the in vitro sensitivity of breast cancer cell lines tofree taxol;

FIG. 10 shows the in vitro testing of free taxol on MDA MB 231 breastcancer xenografts in athymic mice;

FIG. 11 shows the in vitro paclitaxel release from Tx3,Tx4 and Tx5;

FIG. 12 shows the in vitro paclitaxel release from RG504/RG502 blends;

FIG. 13 shows the in vitro paclitaxel release from RG504H/RG502H blends;

FIG. 14 shows the in vitro paclitaxel release from non-H seriescopolymer blends;

FIG. 15 shows the in vitro paclitaxel release from H series copolymerblend;

FIG. 16 shows the in vitro paclitaxel release in the presence of sucrosein the PLGA matrix;

FIG. 17 shows the theoretical plasma concentrations after administrationof dosage forms: (a) intraveous(i.v.)administration and (b) controlledrelease system;

FIG. 18 shows the microsphere morphology of taxol/PLGA: TX Medisorbformulation; Background Count Specifies: 392 particles; mean=6.33 um;median;

FIG. 19 shows the in vitro release kinetics of taxol/PLGA: TX Medisorbformulation; Summary of Taxol Release; (1) Amount of taxol released over10 days (based on supernatant data)=3.41% (308.095 ug); (2) Amount oftaxol released over 10 (based on residual data) 100%−36.13%=63.8%released over 10 days; (3) Time for complete PLGAdegradation=approximately 60-80-days;

FIG. 20 shows the in vitro release kinetics of taxol/PLGA: Tx9formulation; Summary of Taxol Release; (1) Amount of taxol released over10 days (based on supernatant data)=5.70% (525.135 ug; (2) Amount oftaxol released over 10 days (based on residual data) Residual taxol at10 days=67.3% (6207.5 ug) 100 %−67.36%=32.64% released over 10 days;degradation=(3) Time for complete PLGA approximately 30-60 days;

FIG. 21 shows HPLC chromatograms of (A) HPLC grade acetonitrile blank;and (B) 100 ng/ml taxol standard containing NDA, the internal standard;and

FIG. 22 shows the HPLC chromatograms of (A) G6#1 (16 mg/ml; s.c. route;group 1. and (B) G12#1 (16 mg/ml; i.m. route;group 1).

DETAILED DESCRIPTION OF THE INVENTION

The novel compositions described herein are formulated ofchemotherapeutic agent, such as paclitaxel (taxol), doxorubicin,5-fluorouracil, campthothecin, cisplatin, metronidazole campthothecinand combinations, derivatives, or functional equivalents thereof, whichare not water soluble and has poor bioavailability in vivo encapsulatedinto a biocompatible/biodegradable polymeric matrix or in combinationwith hydrophillic agents for use especially in the treatment of breastcancer.

The polymer bound taxol formulations of this invention represent anotherstrategy to achieve prolonged exposure to taxol which appears to be moreadvantageous than infusion regiments. Of greatest utility are thebiodegradable polymers, which include thepoly(lactide-co-glycolide)(PLGA) copolymer. A variety of biodegradablepolymer bound taxol formulations have been developed and have been shownto inhibit tumor growth and angiogenesis in animal models with minimalsystemic toxicity; however, the release kinetics of taxol in thesesystems, which range from 10-25% of the drug released in approximately50 days, are, most likely, not optimal for clinical use(18,17,11,12,19,20,21,22). The advantages of biodegradable polymers as acarrier for taxol include: (1) complete biodegradation, requiring nofollow-up surgery to remove the drug carrier when the drug supply isexhausted (23,24); (2) tissue biocompatibility (25); 3) ease ofadministration via s.c. ir u,n, ubhectuib (23,26,27); (4) controlled,sustained release of the encapsulated drug upon hydrolysis of thepolymer(27,23,28,24);(5) minimization or elimination of systemictoxicity, such as neutropenia 23,24,26,27); and (6) the convenience ofthe biodegradable polymer system itself, in terms of versatility(23,29,24,26).

Surprisingly, our data indicates that we have developed a series oftaxol/PLGA formulations which exhibit different and highly desirablerates of sustained, controlled release of taxol. Due to the sustainedrelease characteristics of these microspheres, taxol/PLGA microsphereformulations are intended as a one-time treatment which sustain releasestaxol from a subcutaneous (s.c) or intramuscular (i.m.) depot.Taxol/PLGA microspheres having a core load of 10%, 20%, 40%, and 50%were prepared via solvent evaporation and were characterized viascanning electron microscopy (SEM), particle sizing, and highperformance liquid chromatography (HPLC). Microsphere morphology ofthese formulations showed intact spheres with an average diameter rangeof 5.69-7.75 um (FIGS. 1,3,5,7). Taxol core loading efficiency of allformulations ranged form 91.9%-95.48%. In vitro release of taxol into 37degrees C PMS/albumin (pH 7.4; 0.4% albumin) over time was calculatedbased on the residual amount of taxol in the microsphere pellet. Resultsshowed: (1)40.19% taxol release in 10 days using a 10% core loadformulation; (2) 71.58% taxol release in 6 days using a 20% core loadformulation (FIG. 4); (3) 48.09% taxol release in 6 days using a 40%core load formulation (FIG. 6); and (4) 39.84% taxol release in 6 daysusing a 50% core load formulation (FIG. 8).

A preliminary toxicity study using placebo PLGA microspheres andtaxol/PLGA microspheres (20% core load formulation) was performed usingC57/black, 6-8 week old, intact female mice. Animals were randomizedinto 12 groups of 4, ear notched, and weighed. Microencapsulated taxoland control polymer were individually resolubilized and injected eithersubcutaneously (s.c.) or intramuscularly (i.m. (inocula volume=50 ul).The right side of the animal received control polymer, and the left sideof the animal received microencapsulated taxol. Each day, animals wereexamined for signs of toxicity. On days 2,4,6, and 8, animals wereweighed, one from each group was sacrificed, and blood was collected forWBC count and for taxol quantitation via HPLC.

Treatment groups were as follows: (1) dose of 0.04 mg/kg using s.c.route; (2) dose of 0.4 mg/kg using s.c. route; (3) dose of 2 mg/kg usings.c. route; (4) dose of 4 mg/kg using s.c. route; (5) dose of 8 mg/kgusing s.c. route; (6) dose of 16 mg/kg using s.c. route; (7) dose of0.04 mg/kg using i.m. route; (8) dose of 0.4 mg/kg using i.m. route; (9)dose of 2 mg/kg using i.m. route; (10) dose of 4 mg/kg using i.m. route;(11) dose of 8 mg/kg using i.m. route; and (12) dose of 16 mg/kg usingi.m. route. Dosage selection was based on the maximum tolerated systemicdose of taxol in mice, which is 16 mg/kg of taxol every 5 days over a 3week period.

Results showed: (1) no signs of toxicity at the injection sites of anyanimal at any time; (2) no signs of weight loss rather, on average, theanimals gained weight; and (3) no appreciable changes in white bloodcell (WBC)count. Taxol concentration in serum samples was too low to bedetectable/quantitable via our taxol extraction and HPLC methods.Additionally, problems experienced with the serum samples-were: (1)protein interference with the taxol peak; (2) moderate to grosshemolysis of the serum samples; and (3) the presence of additional peaksin the chromatogram, which may have been taxol metabolites. Furtherrefinement of our methodologies for taxol extraction from serum and fortrace HPLC analysis, which is currently in progress, will increase thesensitivity of taxol detection and quantitation and will eliminate theseproblems. Since no signs of toxicity were determined, this study isbeing repeated using taxol doses up to 150 mg/kg; however, thispreliminary data suggests that depot administration of taxol viamicrospheres should not cause systemic toxicity.

Applicants have demonstrated that a s.c., or an i.m. injection of taxolencapsulated in a PLGA copolymer is equally as effective as, or perhapsbetter than, conventional systemic taxol therapy for human breast cancertreatment and does not cause the side effects commonly associated withconventional therapy.

Specific Embodiments

Most specifically, the embodiments of this invention are inclusive ofthe following items:

-   1. A controlled release microcapsule pharmaceutical composition of    burst-free, sustained, programmable release of a hydrophobic    bioactive agent over a duration of 24 hours to 100 days, comprising    a hydrophobic bioactive agent and a blend of end-capped and uncapped    biocompatible, biodegradable poly(lactide/glycolide).-   2. The composition of Item 1 wherein the agent is released in an    amount effective to inhibit growth of cancer cells.-   3. The composition of Item 2 wherein the biodegradable    poly(lactide/glycolide) has ratios ranging from 99/1 to 50/50.-   4. The composition of Items 1, 2 or 3 wherein said copolymer has a    molecular weight from 10 to 100 kDa.-   5. The composition of Items 1, 2, 3 or 4 wherein the copolymer is a    blend of hydrophobic end-capped polymer with terminal residues    functionalized as esters and hydrophillic uncapped polymer with    terminal residues existing as carboxylic acids.-   6. The composition of Items 1, 2, 3, 4, or 5 wherein the agent is    selected from the group consisting of paclitaxel, doxorubicin,    5-fluorouracil, camptothecin, cisplatin, metronidazole, and    combinations thereof.-   7. The compositions of Items 1, 2, 3, 4, 5 or 6 further comprising    additional biologically active compounds selected from the group    consisting of chemotherapeutics, antibiotics, antivirals,    antinflammatories, cytokines, immunotoxins, anti-tumor antibodies,    anti-angiogenic agents, anti-edema agents, radiosensitizers, and    combinations thereof.-   8. A method of administering to a patient in need of treatment a    pharmaceutically-effective amount of a hydrophobic bioactive agent    comprising administering the bioactive agent locally to an    infectious area, wherein the agent is incorporated into and    controlled released, burst-free, from a blend of end-capped and    uncapped biocompatible, biodegradable poly(lactide/glycolide) over a    period of 24 hours to 100 days.-   9. The method of Item 8 wherein the bioactive agent is an anticancer    agent.-   10. The method of Item 9 wherein the anticancer agent is selected    from the group consisting of paclitaxol, doxorubicin,    5-fluorouracil, camptothecin, cisplatin, metronidazole, and    combinations thereof.-   11. The method of Item 10 wherein the anticancer agent is    paclitaxol.-   12. The method of Item 8, 9, 10 or 11 wherein the bioactive agent is    administered to the patient prior to the onset of infections.-   13. The method of Item 8, 9, 10 or 11 wherein the bioactive agent is    administered to the patient in need thereof post-infection.-   14. The method of Item 8, 9, 10 or 11 wherein the bioactive agent is    administered intra-muscularly or subcutaneously.-   15. The method of Item 8 further comprising administering radiation    in combination with the composition.-   16. The method of Item 8 further comprising administering with the    bioactive agent additional biologically active compounds selected    from the group consisting of chemotherapeutics, antibiotics,    antivirals, antiinflammatories, cytokines, immunotoxins, antitumor    antibodies, anti-angiogenic agents, anti-edema agents,    radiosensitizers, and combinations thereof.-   17. The method of Item 8 wherein the composition is in the form of    micro-implants and are administered by injection or infusion.-   18. The method of Item 10 wherein the form of cancer being treated    is selected from the group consisting of ovarian, breast, lung,    prostatic, and melanoma, brain tumor cells, and cancer of the    colon-rectum, esophagus, liver, pancreas, and kidney.-   19. A method for inhibiting the proliferation of rapidly    proliferating abnormal mammalian cells, said method comprising    contacting said cells with a cell proliferating inhibiting amount of    an anticancer agent which has been incorporated into and controlled    released, burst-free, from a blend of end-capped and uncapped    biocompatible, biodegradable poly(lactide/glycolide), for a    programmable time sufficient to inhibit said proliferation.

EXAMPLES

The following examples are offered by way of illustration and not by wayof limitation.

Experimental

The abbreviations used herein are defined as follows:

A) Acronym and Symbol Definition

-   ABC=3 amino-9-ethylcarbazole-   CRDA=collaborative research and development agreement-   CT=Chemotherapy-   HPLC=HIGH PERFORMANCE LIQUID CHROMATOGRAPHY-   i.v.=Intra-venous-   i.p.=Intrapariteneal-   i.m.=Intra-muscular-   MDR=Multi-drug resistant-   PBS=Phosphate-buffered saline-   PLGA=Poly(lactide-co-glycolide)-   s.c.=Subcutaneous-   SEM=Scanning electron microscope

Materials and Method

Preliminary Results: Paclitaxel (taxol) is being used as the prototypehydrophobic drug for the development of a PLGA copolymer deliveryvehicle for hydrophobic drugs. Paclitaxel is a diterpene anticanceragent isolated from the bark of the Western Pacific yew, Taxusbrevifolia (FIG. 2) (3). Taxol binds intracellular microtubules,adversely affecting functions critical to cell mitosis via promotion ofabnormal microtubule formation and via stabilization of formedmicrotubules (4,5). Taxol exhibits non-linear pharmacokinetics, in thatthere is a disproportionate relationship between changes in dose andresulting peak plasma concentrations (6). Taxol use is indicated for thetreatment of human breast cancer after failure of combination CT formetastatic disease or upon disease relapse within 6 months of adjuvantCT.

PHASE I. Validation of Methodology for paclitaxel analysis.

A. Accuracy/Precision

1. Experimental Set Up: A 5.0 ug/ml paclitaxel solution was run 10times, and the relative standard deviation percent (RSD%) wascalculated. This validation step was performed on both HPLC instrumentsat U.S. Army Dental Research Attachment, Walter Reed Institute ofResearch (USADRD-WRAIR).

2. Result: The RSD% of System 1 was 1.2131, and the RSDZ of the WatersModular System was 1.592.

B. Detection/Quantitation Limit and Linearity

1. Detection Limit:

-   -   a. Experimental Set-Up: A 0.5 ug/ml taxol solution was serially        diluted to 0.015625 ug/ml. The peak to noise ratio was examined        to determine the detection limit of each HPLC system.    -   b. Result: The detection limit of both HPLC systems was 0.015625        ug/ml.

2. Quantitation Limit/Linearity

-   -   a. Experimental Set-Up: 30 calibration curves from each HPLC run        are being collected for subsequent analysis.        C. Recovery

1. Experimental Set-Up: A known amount of paclitaxel was added toPBS/albumin (the paclitaxel release buffer), and a liquid-liquidextraction of paclitaxel was performed. The concentration of paclitaxelafter extraction was compared to the expected paclitaxel concentrationto verify the efficiency of the extraction methodology.

2. Result: Average percent recovery of paclitaxel after liquid-liquidextraction was 98%.

-   -   D. Sample Solution Stability in PBS/Albumin    -   1. Experimental Set-Up: The stability of paclitaxel in        PBS/albumin was determined over a 6 day period. 4 separate        paclitaxel containing samples were followed over a 6 day period.

2. Result: Average paclitaxel concentration of each sample wascalculated by pooling the 6 days of HPLC data.

-   -   a. Paclitaxel concentration of sample 1: 1.6344+/−0.2895    -   b. Paclitaxel concentration of sample 2: 0.053+/−0.002915    -   c. Paclitaxel concentration of sample 3: 0.0482+/−0.001924    -   d. Paclitaxel concentration of sample 4: 0.0545+/−0.003017    -   E. Specificity/Selectivity

1. Acid/Base Hydrolysis of Paclitaxel

-   -   a. Experimental Set-Up: A 20 ug/ml paclitaxel solution was        exposed to PBS/albumin at the following pH's: 7.4, 10.0, and        2.0. Following a 72 hour exposure, a liquid-liquid extraction of        paclitaxel was performed.    -   b. Result: Acid conditions did not have an effect on paclitaxel,        but basic conditions resulted in paclitaxel degradation.

2. Effect of DH on Paclitaxel Retention

-   -   a. Experimental Set-Up: Mobile phase pH and corresponding        paclitaxel retention time specifics are being recorded after        every HPLC run.    -   F. Robustness        1. Experimental Set-Up: 30 calibration curves from each HPLC run        are being collected for subsequent analysis.    -   G. Effect of Irradiation on Paclitaxex

1. Experimental Set-Up: A 15 mg sample of paclitaxel powder wasirradiated with 20.7-21.4 kGy, a radiation level 10% greater than thestandard 20 kGy target. A comparison between the paclitaxel peak of theirradiated sample was compared to that of an unirradiated sample.

2. Result: Gamma irradiation did not affect the paclitaxel peak.

-   -   H. Comparison Between Liquid/Liquid and Solid Phase Extraction

1. Experimental Set Up: Liquid/liquid extraction of paclitaxel is highlytime-consuming and labor intensive, therefore, an alternative method forpaclitaxel extraction, solid phase extraction, was recently examined interms of extraction efficiency. Liquid/liquid extraction and solid phaseextraction were performed on 3 separate 2 ug/ml paclitaxel solutions.

2. Result: Area comparisons were follows:

-   -   a. 2 ug/ml paclitaxel solution (no extraction): 52,230+/−3,244    -   b. 2 ug/ml paclitaxel solution (liquid/liquid extraction):        48,648+/−2,571    -   c. 2 ug/ml paclitalex solution solid phase extraction):        59,346+/−4,962    -   d. Solid phase extraction of paclitaxel will replace        liquid/liquid extraction of paclitaxel.    -   I. Photodiode Array (PDA) Detection of Paclitaxel

1. Experimental Set-Up: A paclitaxel standard in acetonitrile/d. waterwas prepared and run through the PDA detector. This result was used forthe Millennium's PDA spectrum purity analysis as the reference spectrato compare against 3 in vitro samples, extracted by liquid/liquidextraction, containing a known amount of paclitaxel. The purity and thethreshold angle between the paclitaxel samples and the paclitaxelstandard were calculated. These calculated numbers correspond to thepurity of the paclitaxel peak that is being analyzed via paclitaxel'sHPLC methodology.

2. Result: For each comparison, the purity angle was less than thethreshold angle, indicating that the paclitaxel peak being analyzed viaHPLC is pure.

PHASE II: Production and Analysis of Paclitaxel/PLGA Microspheres

-   -   A. Preliminary Screening of Paclitaxel/PLGA Microsheres    -   1. Purpose: Preliminary screen of a series of paclitaxel        microspheres produced using a variety of PLGA copolymers and of        external phase stir rates.        -   a. Copolymers Examined:        -   High Molecular Weight PLGA: RG504, a non-H series copolymer,            and RG504H, an H-series copolymer.        -   Low Molecular Weight PLGA: RG502 and RG502H            Rationale for Screening Different Molecular Weight            Copolymers: As the molecular weight of the copolymer            decreases, drug release rate increases.            Rationale for Screening H vs. Non-H Series            Copolymers: H-series copolymers hydrate faster than the            non-H series copolymers due to the presence of carboxylic            acid end chains. Quicker hydration of the copolymer results            in an accelerated drug release rate.        -   b. External Phase Stir Rates Examined: 250, 500, and 960            rpm.            Rationale for Examining Different Stir Rates: As the stir            rate increases, smaller microspheres result in an            accelerated drug release rate.

2. Result

-   -   a. Paclitaxel Core Loads Paclitaxel-loaded microspheres were        dissolved in acetonitrile/d. water and then run on the HPLC        against a set of paclitaxel-containing standards (Table 1).

For- Stir Rate Paclitaxel % Core % Theoretcial % Loading PaclitaxelPLGA, mg ymulation (rpm) (ug/ml) Load CL CL Efficiency Mg and Type Tx03250 45.388 4.407 5 88.131 10 190RG504 Tx04 250 97.905 9.324 10 93.243 20180RG504 Tx05 250 176.104 16.772 20 83.859 40 160RG104 Tx07 960 96.4079.010 10 90.100 20 180RG504 Tx08 960 166.076 15.969 20 79.844 40160RG504 Tx09 960 100.628 9.676 10 96.758 20 180RG504H Tx10 960 185.82917.206 20 86.032 40 160RG504H Tx11 960 91.340 8.955 10 89.549 20180RG502 Tx12 960 173.742 16.706 20 83.530 40 160RG502 Tx13 500 85.1378.108 9.090 89.191 20 200RG504 Tx14 500 144.042 13.850 16.667 83.101 40200RG504 Tx15 500 83.251 7.929 9.090 87.215 20 200RG504H Tx16 500153.061 14.717 16.667 88.304 40 200RG504H Tx17 500 89.999 8.490 9.09093.395 20 200RG502 Tx18 500 157.295 14.700 16.667 88.203 40 200RG502Tx19 250 82.756 7.882 10 78.815 20 180RG504 Tx20 250 163.937 16.394 2081.969 40 160RG504 Tx21 250 75.590 7.268 10 72.683 20 180RG504H Tx22 250172.609 16.439 20 82.195 40 160RG504H Tx23 250 87.821 8.364 10 83.639 20180RG502H Tx24 250 168.144 16.168 20 80.838 40 160RG502 Tx25 960 100.5229.573 10 95.735 20 180RG503 Tx26 960 194.583 18.710 20 93.550 40160RG503 Tx27 960 95.110 9.324 10 93.245 20 180RG503H Tx28 960 185.92918.593 20 92.965 40 160RG503H Tx29 960 103.769 10.173 10 101.734 20180RG502H Tx30 960 191.514 18.415 20 92.074 40 160RG502H Tx31 500 90.8238.992 10 89.924 20 180RG503 Tx32 500 183.843 17.849 20 89.244 40160RG503 Tx33 500 97.025 9.240 10 92.405 20 180RG503 Tx34 500 193.37518.417 20 92.083 40 160RG503H Tx35 500 97.559 9.565 10 95.646 20180RG502H Tx36 500 190.431 18.488 20 92.442 40 160RG502H Tx37 250 90.4978.872 10 88.723 20 180RG503 Tx38 250 151.375 14.841 20 74.203 40160RG503 Tx39 250 94.872 9.211 10 92.108 20 180RG503H Tx40 250 193.06819.307 20 96.534 40 160RG503H Tx41 250 100.750 9.782 10 97.816 20180RG502H Tx42 250 180.313 18.031 20 90.157 40 160RG502H PaclitaxelLoading Efficiency: 72-96%

b. Scanning Electron Microscope Results:

-   -   The morphology of paclitaxel-loaded microspheres was examined        via SRM.    -   RG504 Copolymer: Intact microspheres, with an average diameter        of 100 um.    -   RG504H Copolymer: Intact microspheres, with an average diameter        of 100 um.    -   RG503 Copolymer: Intact microspheres, with an average diameter        of 100 um. Morphologic damage, including cracked, misshapen,        hollow, and dented microspheres, was noted.    -   RG503H: Intact microspheres, with an average diameter of 100 um.        Morphologic damage was noted.    -   RH502: No intact microspheres were present.    -   RG502H: No intact microspheres were present.

c. In vitro Paclitaxel Release

-   -   Experimental Set-Up: 10 mg of paclitaxel-loaded microspheres        were suspended in 50 ml PBS/albumin (0.2% mass) and placed in a        37 degree Celsius shaking water bath. At specified time        intervals, 48 ml of the supernatant was removed for subsequent        HPLC analysis and the sample was replenished with 48 ml of fresh        buffer. The 50 ml volume and the timing of the sampling ensure        that sink conditions are met.    -   In vitro Paclitaxel Release From Formulations Tx3. Tx4. and Tx5        (FIG. 11):    -   (a) Paclitaxel-loaded microspheres containing RG503(non-H and H)        and RG502(non-H and H), as opposed to those containing        RG504(non-H and H), showed structural damage; therefore, only        RG504(non-H and H) containing microspheres were selected for        subsequent analysis.    -   (b) All formulations which consisted of intact, undamaged        microspheres averaged microsphere diameters of 100 um;        therefore, a subset of these formulations, Tx3, Tx4, and Tx5,        were selected for subsequent release analysis.

3. Conclusions

-   -   a. Paclitaxel was efficiently encapsulated in PLGA using solvent        evaporation methodology.    -   b. As the molecular weight of the polymer decreased,        paclitaxel-loaded microspheres lost structural stability.    -   c. The stir rate effect, in terms of microsphere size, was not        highly evident due to the molecular weight of the copolymer.        Using high molecular weight copolymer, RG504 and RG504H,        resulted in large microspheres regardless of the applied        external phase stir rate.    -   d. Paclitaxel release from RG504 was slow due to the large size        of the microspheres.        B. Paclitaxel Formulations Consisting of RG504 (non-H and        H)/RG502 9non-H and H) Blends

1. Purpose and Rationale: To improve the structural stability ofRG502(non-H and H)-containing paclitaxel microspheres and to increasethe paclitaxel release rate via blending RG504(non-H and H), the highmolecular weight copolymer, with RG502(non-H and H), the low molecularweight copolymer. The rationale for this approach is that RG504(non-Hand H) will provide structural stability to the microsphere and thatRG502(non-H and H) will allow for faster hydration of the microspheres.Faster hydration results in accelerated drug release.

2. System Parameters Examined: Combinations of either RG504 and RG502 orRG504H and RG502H. External phase stir rate was held constant at 1000rpm in order to determine the effect of microsphere composition, interms of the ratio of RG504(non-H and H) to RG502(non-H and H), uponpaclitaxel release.

TABLE 2 Paclitaxcl Core Loads For- Paclitaxel % Core % Theoretical %loading Paclitaxel, PLGA, mg mulation (ug/ml) Load CL efficiency mg andtype A 104.742 9.88 10 96.814 20 180 5% RG504/95% RG502 B 84.414 8.44110 84.415 20 180 10% RG504/90% RG502 C 94.004 8.953 10 89.528 20 180 50%RB504/50% RG502 D 100.560 9.866 10 98.656 20 180 75% RG504/25% RG502 E93.288 9.057 10 90.570 20 180 95% RG504/5% RG502 F 100.667 9.680 1096.795 20 180 90% RG504/10% RG502 G 89.189 8.494 10 84.941 20 180 25%RG504/75% RG502 A1 95.627 9.375 10 93.752 20 180 5% RG504H/95% RG502H B192.768 9.185 10 91.849 20 180 10% RG504H/90% RG502H C1 100.761 9.763 1097.826 20 180 D1 104.187 10.018 10 100.018 20 180 50% RG504H/50% RG502HE1 93.277 9.235 10 92.353 20 180 95% RG504H/%5% RG502H F1 92.469 9.24910 92.480 20 180 90% RG504H/10% RG502H G1 96.491 9.849 10 96.491 20 18025% RG504H/75% RG502H Paclitaxel Loading Efficiency: 84-99%

b. SEM Results:

-   -   RG504/RG502 Blends:    -   TxA: Intact microspheres, with an average diameter of 87 um.    -   TxB: Intact microspheres, with an average diameter of 120 um.    -   TxC: Intact microspheres, with an average diameter of 144 um.    -   TxD: Intact microspheres, with an average diameter of 212 um.    -   TxE: Intact microspheres, with an average diameter of 244 um.    -   TxF: Intact microspheres, with an average diameter of 220 um.    -   TxG: Intact microspheres, with an average diameter of 116 um.        RG504H/RG502H Blends    -   TxA1: Intact microspheres, with an average diameter of 63 um.    -   TxB1: Intact microspheres, with an average diameter of 83 um.

TxC1: Intact microspheres, with an average diameter of 108 um.

-   -   TxD1: Intact microspheres, with an average diameter of 112 um.    -   TxE1: Intact microspheres, with an average diameter of 276 um.    -   TxF1: Intact microspheres, with an average diameter of 268 um.    -   TxG1: Intact microspheres, with an average diameter of 140 um.

4. Conclusions

-   -   a. Paclitaxel loading efficiency was high using the copolymer        blend methodology.    -   b. The structural stability of RG502(non-H and H)-containing        paclitaxel microspheres was optimal with the copolymer blend        methodology.    -   c. As the concentration of RG504, high molecular weight        copolymer, increased, the size of the microsphere increased.        This relationship held true for H series and non-H series        copolymers.    -   d. As the concentration of RG502(non-H and H)increased, the        paclitaxel release rate increased.    -   e. In comparing paclitaxel release rates at the end of the        release period, paclitaxel formulations containing the H-series        copolymers released paclitaxel at a rate 3-10 times greater than        those containing the non-H series copolymers; therefore, the        H-series copolymers significantly increased paclitaxel release        rates.    -   f. The size of the microsphere was affected by the molecular        weight of the copolymer, with predominantly H-series containing        paclitaxel formulations having the smallest microspheres.    -   g. Smaller microspheres which contained a higher percentage of        RG502(non-H and H) exhibited paclitaxel release rates faster        than larger microspheres which contained a higher percentage of        RG504(non-H and H).        C. Manipulation of System Parameters at the Microsphere        Production Level to Further Increase Paclitaxel Release Rates

1. Purpose: The primary goal of this set of formulations was to furtherincrease paclitaxel release rates by further manipulating systemparameters at the microsphere production level.

2. System Parameters:

-   -   a. Sonication Stew: The paclitaxel/PLGA solution was sonicated        with an aliquot of the external phase solution to form an        emulsion. This emulsion resulted in the formation of small        droplets of taxol and PLGA; therefore, smaller microspheres, on        the order of 2-10 um in diameter, were produced.    -   b. Cooling Step: During the first half hour of stirring the        paclitaxel PLGA in the external phase, the temperature of the        external phase was maintained at 15 Celsius. This cooling        facilitated the formation of smaller microspheres by minimizing        the early aggregation of the small and numerous droplets of        paclitaxel/PLGA.    -   c. External Phase Stir Rates: range of 250-1800    -   d. Formulation Specifics: 10% paclitaxel core loads (20 mg        paclitaxel) with PLGA copolymer blend consisting of 90% RG502        (non-H and H) (162 mg) and 10% RG504 (non-H and H) (18 mg).        3. Results

TABLE 3 Paclitaxel Core Loads For- Stir Number Paclitaxel % Core %Theoretical % loading PLGA mulation Rate of Bursts (ug/ml) Load CLefficency Type Tx54 1800 4 68.315 6.737 10 67.372 Non-H Tx55 1800 476.381 7.43 10 74.300 H Tx56 1000 3 50.225 4.829 10 48.293 Non-H Tx571000 3 45.268 4.356 10 43.526 H Tx58 500 3 51.874 5.178 10 51.770 Non-HTx59 500 3 48.738 4.677 10 46.774 H Tx60 700 3 43.282 4.174 10 41.738Non-H Tx61 700 3 52.980 5.596 10 55.960 H Tx62 250 3 97.377 11.248 10112.480 Non-H Tx63 250 3 106.140 11.997 10 119.970 H Tx64 500 1 106.12512.370 10 123.700 H Tx65 500 0-vortex 109.854 12.204 10 122.040 H Tx66500 3 103.761 11.811 10 118.110 H Paclitaxel Loading Efficiency 420100%

b. SEM Results

-   -   Tx54: Intact microspheres, with an average diameter of 2.5 um.    -   Tx55: Intact microspheres, with an average diameter of 2.0 um.    -   Tx56: Intact microspheres, with an average diameter of 9.6 um.    -   Tx57: Intact microspheres, with an average diameter of 3.0 um.    -   Tx58: Intact microspheres, with an average diameter of 7.0 um.    -   Tx59: Intact microspheres, with an average diameter of 11.0 um.    -   Tx60: Intact microspheres, with an average diameter of 40.0 um.    -   Tx61: Intact microspheres, with an average diameter of 49 um.    -   Tx62: Intact microspheres, with an average diameter of 32 um.    -   Tx63: Intact microspheres, with an average diameter of 12 um.    -   Tx64: Intact microspheres, with an average diameter of 18 um.    -   Tx65: Intact microspheres, with an average diameter of 17 um.    -   Tx66: Intact microspheres, with an average diameter of 26 um.    -   Advanced Development of Selected Paclitaxel/PLGA Formulations:        Tx57. Tx9 and TxMedi

a. preparation of Tx57

-   -   1) Set water bath to 15 C.    -   2) Prepare 1% poly-vinyl alcohol (PVA) in distilled water—500 ml    -   3) Co-dissolve paclitaxel powder and PLGA in 3.5 g methylene        chloride (CH2C12)    -   4) Add this solution to 25 ml of 1% PVA in CH2C12-saturated        distilled water    -   5) Homogenize five seconds (approx. 10,000 rpm)    -   6) Vortex 5 seconds    -   7) Add homogenized solution to the 500 ml PVA/water    -   8) Spin at 15 C. for ½ hour—stir rate 500 rpm    -   9) Spin at 15 C. for 4 hours—stir rate 500 rpm    -   10) Filter/wash spheres/vacuum dry at room temp overnight.

b. Preparation of Tx9 and TxMedi

-   -   1) Set water bath to 15 C.    -   2) Prepare 1% PVA in distilled water—500 ml.    -   3) Co-dissolve paclitaxel powder and PLGA in 8.1 gCH2C12    -   4) Add this solution to 25 ml of 1% PVA in CH2C12-saturated        distilled water    -   5) Vortex 5 seconds until consistency is milky/frothy.    -   6) Add vortexed solution to 500 ml of stirring PVA/water.    -   7) Spin 15 C. for ½ hours—stir rate 500 rpm    -   8) Spin 15 C. for 4 hours—stir rate 500 rpm    -   9) Filter/wash spheres/vacuum dry at room temp overnight

C. Characterization of Tx57, Tx9, and TxMedi

6. Advanced Development of Selected Paclitaxel/PLGA Formulations: Tx20%.Tx40%. and Tx50%

a. preparation of Microspheres

-   -   1) Set water bath to 15 C. (run for ½ hour at this temp before        starting).    -   2) Prepare 1% PVA in distilled water—500 ml    -   3) Co-dissolve paclitaxel powder and PLGA in 3.5 g CH2C12    -   4) Add this solution to 25 ml of 1% PVA/CH2C12 saturated        distilled water    -   5) Homogenize (10,000 rpm/30 seconds in 50 ml centrifuge tube)    -   6) Add homogenized solution to the 500 ml PVA/water    -   7) Stir ½ hour at 15 C.—stir rate=650 rpm    -   8) Stir 4 hours at 25 C.—stir rate=650 rpm    -   9) Filter/wash spheres/vacuum dry overnight        b. Characterization of Tx20%. Tx40%. and Tx50%

In FIGS. 2,3,6, and 8 the amount of taxol released based on residualcore load(res) data is more accurate than the amount released based onsupernatant (sup) data.

-   -   d. Conclusions    -   1. The additional production level steps lowered paclitaxel        loading efficiency because, most likely, these steps required        the transfer of the paclitaxel/PLGA solution to multiple        beakers.    -   2. Addition of the previously mentioned steps resulted in        significantly smaller microspheres, as compared to other batches        of microspheres produced via methodology described in phase II,        section A and B.    -   3. Sonication and cooling steps resulted in the production of        microspheres on the order of 2-10 um in diameter. Average        microsphere diameter of microsphere populations was inversely        proportional to the external phase stir rate; however,        microsphere diameter ranges were highly variable. This variation        was not as pronounced in microsphere populations produced via        methodology described in phase II, section A and B.    -   4. Intra-formulation range of microsphere diameters was great;        therefore, overall patterns of paclitaxel release in response to        the specific system parameters were slightly masked. Despite        this technically, the general pattern of paclitaxel release was        that H-series formulations released paclitaxel faster than non-H        series formulations.    -   5. Overall, the system parameters which accelerate paclitaxel        release from PLGA are (a) the use of low molecular weight PLGA        are: (a) the use of low molecular weight PLGA copolymer; (b) the        use of high external phase stir rates; and (c) the sonication of        the taxol/PLGA solution before external phase stirring.

D. Addition Of Sucrose into the Copolymer Matrix to Accelerate

Paclitaxel Release

-   -   1. Purpose and Rationale: Incorporate an additive into the        copolymer matrix to accelerate paclitaxel release. The rationale        of this approach is that sucrose will immediately hydrolyze and        create channels with the microsphere to allow for quicker        hydration; thus, paclitaxel release should be significantly        accelerated.

2. System Parameters:

-   -   a. Procedure: solvent extraction    -   b. Stir Rate: 700 rpm for each batch    -   c. Homogenization: 50% power, 30 second burst    -   d. Polymer used RG501H    -   e. Additive Used: sucrose (1.266%) or no sucrose    -   f. Theoretical Paclitaxel Core Load: 10.127%

3. Results

-   -   a. Paclitaxel Cor e J,oads    -   Batch 1 sucrose 10.724%    -   Batch 2 (no sucrose) 10.398%

b. SEM Results:

-   -   Batch 1: Intact microspheres, with an average diameter of 6 um.    -   Batch 2: Intact microspheres, with an average diameter of 5 um.    -   C. In Vitro Paclitaxel Release (FIG. 8)

d. Conclusions

-   -   1. Sucrose within the matrix of the PLGA matrix accelerates        paclitaxel release in comparison to release in the absence of        sucrose.    -   2. Paclitaxel release in the presence of sucrose was similar to        other paclitaxel/PLGA formulations described above (compare FIG.        8 to FIG. 6). A potential reason for this may be that the        percent sucrose within the PLGA matrix, which was 1.27% was not        high enough to cause a significant increase in paclitaxel        release.    -   3. Additional paclitaxel/PLGA formulations containing more than        1% sucrose within the copolymer matrix are in the process of        being prepared.        B. The Advantage of the Invention Over Presently Known Devices        Systems, or Process.

Encapsulation of hydrophobic drugs in PLGA copolymer offers numerousadvantages over standard hydrophobic drug administration. The advantagesof biodegradable PLGA copolymer as a carrier for hydrophobic drugsinclude: (1) complete biodegradation, requiring no follow-up surgery toremove the drug carrier when the drug supply is exhausted (2,7); (2)biocompatibility(8); (3) ease of administration, in that anticancerdrugs incorporated into biodegradable polymers can be administered viasubcutaneous(s.c.) or intra-muscular(i.m.) injection (2,9,10); and (4)the convenience of the biodegradable copolymer system itself, in termsof versatility and cost (2,7,9,11).

Most importantly, hydrophobic drugs incorporated into PLGA copolymerwould allow for controlled release of the drug, as evidenced by drugrelease from previously reported lactide/glycolide drug delivery systems(2,7,10,12,13,14,15,16).

A variety of chemotherapy (CT) drug delivery systems have been reportedin the current literature; however, problems of poor, limited drugrelease from the drug carrier and of burst phenomenon are apparent. CTdrugs are highly hydrophobic in nature; thus, CT drugs belong to theclass of drugs which are illustrative of this invention.

Taxol encapsulated into liposomes prepared from phosphatidylglycerol andphosphatidylcholine were tested in mice bearing colon-26, a taxolresistant murine adenocarcinoma (17,18). Taxol encapsulated intopoly-(e-caprolactone)(19) and into microspheres of ethylene vinylacetate copolymer and poly(d,1-lactic acid) (20) have been tested fortheir ability to inhibit angiogenesis. Taxol encapsulated intonanocapsules composed of a polymeric wall of poly lactic acid have beentested in murine leukemia models; however, toxicity of the carrier wasdemonstrated (21). Overall, these studies show tumor growth inhibitionand angiogenesis inhibition; however, the release kinetics of taxol inthese systems, which range form 10-25% of the drug released inapproximately 50 days, may be sub-optimal, in terms of taxol dosages tobe used clinically.

Cisplatin encapsulated in PLGA has been developed for local,intraperitoneal (i.p.) administration in the context of ovarian cancer(22) In vivo data indicated that i.p. administration of cisplatin/PLGA,as opposed to i.p. administration of an aqueous solution of cisplatin,increased the mean survival time of tumor-bearing rats; however, thisformulation was not a stable cisplatin-releasing dosage form because ofthe burst phenomenon.

The conditions under which we encapsulate hydrophobic drugs (see sectionA) are specifically designed to overcome the limitations of the CT drugdelivery systems described above.

C. Discussion of Problems Which the Invention is Designed to Solve.

The pharmacokinetics of systemically administered drugs is, typically,problematic in terms of efficacy and of toxicity. Standard i.v.administration of hydrophobic drugs, such as paclitaxel, results inalternating periods of overdose and drug inefficacy, whileadministration of controlled release drug formulations eliminates thosepeaks and valleys and provides a more constant systemic drugconcentration over time (FIG. 9) (23) Systemic i.v. administration oftaxol is the primary means of delivering potent CT drugs to cancerpatients. Systemic CT is highly effective in cancer treatment, in termsof additional years of life gained as a result of therapy; however,there are many problems associated with systemic CT. CT drugs are highlycytotoxic (24), and, typically, large doses of CT drugs are needed toproduce an optimal therapeutic response (24,25); therefore, CT drugshave a low therapeutic index (24).

Side effects commonly seen with standard CT dosages include: nausea,vomiting, fever, weight changes, hot flashes, musculoskeletal pain,alopecia, dermatitis, conjunctivitis, general malaise, and immunedysfunction (26,27). An additional side effect seen in patients treatedwith taxol is a severe hypersensitivity reaction due to Cremophor EL,taxol's solubilizing agent (17). Often, patients become so ill from CTthat they have to be removed from treatment regimens (28). Theconsequence of this removal from therapy is fluctuating CT drug levels,which equate to decreased efficacy of CT drugs.

The PLGA copolymer technology discussed in this disclosure is designedto bypass the problematic pharmacokinetics of systemically administereddrugs and the systemic toxicity typically associated with this mode ofadministration. The in vivo pharmacokinetics of a PLGA/drug formulation,in which there is controlled release of the drug from the PLGAcopolymer, will, most likely, eliminate systemic toxicity (FIG. 9). Attime 0 of standard i.v. drug therapy, a toxic amount of drug isdelivered systemically. This toxic dose is a shock to the patient'sbiologic system, causing significant toxic side effects within a shortperiod of time. This toxic drug level then rapidly drops to one ofminimal effectiveness. This pattern ultimately results in a shortexposure time of the biologic system to a therapeutic level of drug.

Drug release from a controlled release system is smooth and constant,with an extended period of time that the systemic concentration of thedrug is at a therapeutic level. This extension is highly advantageousbecause it provides a more effective drug regimen. An additional medicalconcern when administering CT drug is that of drug interaction problems.Standard taxol CT requires pre-medication with corticosteroids,antihistamines, and histamine H2 receptor antagonists because of thetoxicity of Cremophor EL. This pre-medication increases the potentialfor drug interactions that can affect taxol's pharmacokinetics andpharmacodynamics. As a PLGA copolymer formulation of this drug requiresPLGA copolymer powder and taxol powder only, Cremophor EL can beeliminated from taxol CT; thus, taxol tolerability should be enhanced.

Given that hydrophobic drugs, such as CT drugs, are highly problematicunder standard conditions of systemic administration, PLGA formulationsfor this class of drugs which minimize or eliminate systemic toxicityvia controlled drug release will lead to more effective drug regimensand to a more efficient and economical use of this class of drugs.

D. List all Known and Other Possible Uses for the Invention

PLGA copolymer technology will provide clinicians with an alternative tosystemic administration of hydrophobic drugs, such as taxol doxorubicin,5-fluorouracil camptothecis, cisplatin, and metronidazole, which areproblematic in terms of systemic toxicity complications. Thisalternative is that of a a.s.c./i.m. depot which control releaseshydrophobic drugs. The advantages of this depot system in terms ofhydrophobic drug delivery are: (1) improved patient compliance, as thenumber of drug dosings are decreased because the depot contains anamount of drug equivalent to multiple doses; (2) isolation depot fromthe tissue via its incorporation in PLGA thus reducing the drugconcentration exposed to the one time and decreasing the chance oftissue injury of the drug copolymer, tissue at any at the depot site;(3) controlled drug release, which may allow for increased dosages ofhydrophobic drugs to be administered without systemic toxicitycomplications. In terms of specific clinical applications of thistechnology, hydrophobic drug/PLGA formulations are envisioned to play arole in the treatment regiment of cancer and of infection.

Administration of paclitaxel/PLGA microspheres will offer a safer and amore effective means of delivering taxol to breast cancer patients thanstandard i.v. administration. Systemic taxol CT is now approved by theFederal Drug Administration (FDA) for the treatment of breast cancerafter failure of combination CT for metastatic disease or after relapsewithin 6 months of adjuvant CT. The proposed paclitaxel/PLGA formulationwill potentially, be used under the specific conditions as analternative to systemic taxol CT. Additionally, the increasedtolerability and efficacy of paclitaxel encapsulated in PLGA copolymerwill, potentially, make this formulation an exemplary first-line CTagent for the treatment of breast cancer.

An expanded use of the proposed paclitaxel/PLGA formulation isenvisioned Paclitaxel/PLGA may potentially be therapeutic for cancersother than breast cancer which are currently being treated with taxol CTregiments. Due to the increased tolerability of paclitaxel/PLGA,paclitaxel may be considered as a component of first-line CT regimentsfor tumors or various origin.

Additionally, the process of microencapsulating paclitaxel in PLGAcopolymer is, most likely, capable of being applied to themicroencapsulation of hydrophobic drugs other than paclitaxel. Themicroencapsulation technology used to prepare paclitaxel/PLGAmicrospheres is not specific to paclitaxel only, as it is a generalchemical process. The advantages of microencapsulating paclitaxel inPLGA copolymers, in terms of improved tolerability and efficacy, areones that can, most likely, be applied to any CT drug whose standardtreatment results in undesirable systemic toxicity. Potential CT drugsto benefit from the PLGA copolymer technology described in thisdisclosure include: metronidazole, cisplatin, doxorubicin, camptothecin,and Sfluorouracil.

Moreover, this invention relates to and contemplates the use of thenovel PLGA copolymer technology as a delivery system for bioactiveagents such as heart drugs, like Capoten, Prinivil/Prinzidel; Vasotec,Pepcid,Mevacor, Antidepressents, like Prozac; arthritic drugs, likeNaprosyn; antihistimines, like Claritin, Deconamine; anti-ulcer drugs,like Prilosec, Zantac; antiherpes drugs like Zovirax; Aids treatmentdrugs like, Crixivan, AZT; drugs for treating osteoporisis, like Esamax;blood pressure drugs; and drugs for the treatment of anxiety, diabetes,insomnia, alzheimer disease, migraine headaches, vitamins and dietregulating drugs, the active ingredient in each, derivatives andcombinations thereof, or in combination with other bioactive agents.

E. List the Features of the Invention that are Believed to be Novel

Novel Features of Described Hydrophobic Drug/PLGA Formulations:

1. Hydrophobic drug/PLGA administration via the s.c. or i.m. route, aformulation which has the characteristics of: (1) depot formulation; (2)controlled drug release.

2. Elimination or significant decrease in the systemic toxicity ofhydrophobic drugs, such as paclitaxel, cisplatin, doxorubicin,5-fluorouracil, camptothecin, and metronidazole, due to thepharmacokinetics of controlled release drug formulations.

A) Produced sufficient quantities of our previously developed taxol/PLGAformulations for in vitro and in vivo testing.

B) Conduct in vitro analysis of the efficacy of taxol/PLGA using a taxolsensitive uterine sarcoma cell line and various human breast cancer celllines.

C) Using a nude mouse xenograft model:

1) Establish a maximum tolerated dose for the series of taxol/PLGAformulations.

2) Compare the in vivo tumoricidal effect of taxol/PLGA to that ofconventional administration of free taxol.

Methods Practiced

A) Production of taxol/PLGA Microspheres

1) Taxol/PLGA microspheres were prepared via solvent evaporation.

2) Core load, the percentage of taxol captured in the PLGA copolymer,was determined via HPLC using a pentylfluoryl phenyl column.

3) Morphology of taxol/PLGA microspheres were examined via SEM. Particlesize distribution of the taxol/PLGA microspheres were calculated usingthe Accusizer Model 770 Single Particle Optical Sizer.

4) Taxol release from the PLGA copolymer were determined via taxolextraction using a SPE column and subsequent HPLC analysis.

B) In Vitro Analysis of the Efficacy of Taxol/PLGA Microspheres

1) Commercial cell lines used to test microspheres: ATCC CRL-1902;primary breast carcinoma, human; ATCC CRL-1897; breast carcinoma, human;ATCC CRL-1976; taxol sensitive uterine sarcoma, human. Institutionalcell lines to be used to test microspheres: MDA MB 435 cells, MDA MB 231cells, and MCF-7 cells, all of which are breast cancer cell lines.

2) Cell lines were subcultured to expand the line for subsequent invitro testing.

3) Cells from each cell line will be tested in exponential growth phase.24 hours post plating, wells will be treated with media only, media+freetaxol (solubilized in Cremophor EL), or media+taxol/PLGA microspheres.Cells will be harvested 0,24,48, and 72 hours post taxol treatment forsubsequent in vitro analysis. Taxol treatment of breast cancer celllines is appropriate methodology to study the in vitro effect of taxol,as taxol/Cremophor EL treatment of various breast cancer cell lines hasbeen shown to have anti-tumor effect, in terms of cytotoxicity (FIG. 9).

4) In vitro assays performed at each time point:

-   -   a) Proliferation of breast cancer cells: indices of        proliferation of taxol treated breast cancer cell lines were        determined using the MTT Assay Cell Titer Kit, a commercially        available cell proliferation kit (Prometa, Madison, Wis.).    -   b) Cytocentrifuge slides of cells were stained using the        Papanicolau staining procedure and will be examined for        chromatin condensation and/or fragmented nuclei. Image analysis,        examining parameters such as cell area, nuclear area, and        cytoplasmic area, were performed using the Zeiss KT-400 image        analysis system as the means of quantitating histologic data. As        a separate staining procedure, cells will be stained with        anti-cx-tubulin (Sigma, St. Louis, Mo.), a stain which binds the        microtubule protein, tubulin. Staining was visualized using an        AEC staining kit. Microtubular changes was examined via image        analysis as the means of quantitating immunohistologic data.        C) In Vivo Analysis of the Efficacy of Taxol/PLGA Microspheres

1) The efficacy of taxol/PLGA microspheres was evaluated using a nudemouse xenograft model. This model has successfully and safely been usedat the Lomardi Cancer Research Center, as evidenced by taxol/CremophorEl treatment of athymic nude mice carrying MDA 231 human breast cancerxenografts (FIG. 10). Briefly, mice will be inoculated s.c. into themammary fat pads at 2 sites per mouse with 1×10 to 2×10 MDA 231 humanbreast cancer cells which have been MAP tested. Taxol treatment wasbegun 1-30 days post cell inoculation. Saline was the injection vehiclefor the microspheres, and Cremophor EL was the injection vehicle for thefree taxol. The taxol/Cremophor EL treatment schedule was approximately3 weeks duration on a 5 day basis. The taxol/PLGA treatment scheduleconsisted of a one dose, i.m. or s.c. injection for the approximate 3week duration.

2) Animals were sacrificed at specified time points during the treatmentregimen.

3) Testing at animal-harvest time points:

-   -   a) Determination of the concentration of taxol in the serum and        at the tumor site of taxol/PLGA treated animals via        liquid-liquid extraction using diethyl ether and analysis of the        dried organic phase via HPLC. Additionally, the HPLC methodology        incorporates the use of an internal standard,        N-nitrosodiphenylamine. Comparison of taxol concentration in        response to taxol microsphere treatment to that in response to        free taxol was made.    -   b) Evaluation of local toxicity of taxol/PLGA microspheres via        examination of the injection sites and biopsy of the sites when        necessary.    -   c) Evaluation of systemic toxicity of taxol/PLGA microspheres        via a complete blood cell count and recorded weight changes was        done. Comparison between taxol microsphere treated and free        taxol treated animals was made.

Spore Animal Experiment #20:

Objective: Conduct toxicity study on microencapsualted taxol. Study willbe done using C57/black, 6-8 week old, intact female mice. Animals willbe injected (inocula volume=50 ul) on Day 0 either subcutaneously orintramuscularly. Each day they will be examined for signs of toxicity.On days 2,4,6 & 8 animals will be weighted and one from each group willbe sacrificed. A WBC will be done and serum collected. If any signs oftoxicity are seen at the sight of injection, the site will be excised,fixed in 10% formalin, paraffin embedded and H&E's made.

Procedure:

Day 1—Randomize mice into 12 groups of 4 and ear notched & weigh

Day 9—Resolubilize microencapsulated taxol and control polymer—Weightanimals and inject. RIGHT side will receive polymer control and LEFTside will receive encapsulated taxol

Day 11—all animals

-   -   Take on animal from each group, collect blood samples for WBC        and serum. Check injection site for signs of toxicity. If signs        exist, collect specimens.        Day 13—Day 11 procedure        Day 15—procedure        Day 17—procedure    -   *Everyday animals will be examined once in the morning and once        in the evening for any signs of toxicity. If we see or deem        morbidity inevitable, blood and specimens will be collected.

Treatment Groups: *Group # Dose Route 1 0.04 mg/kg Subcut 2 0.4 mg/kgsubcut 3 2 mg/kg subcut 4 4 mg/kg subcut 5 8 mg/kg subcut 6 16 mg/kgsubcut 7 0.04 mg/kg IM 8 0.4 mg/kg IM 9 2 mg/kg IM 10 4 mg/kg IM 11 8mg/kg IM 12 16 mg/kg IM *Group # corresponds to Cage # on spread sheet,(Table 4)Notes:

Injection site—no signs of animal at any time. This is true control,taxol, subq or IM,

Weight—no signs of weight loss, on average the animals gained weight,

WBC—no appreciable change in white blood cell count detected,

Conclusion:

-   -   Since no signs of toxicity were determined, repeat study with        high doses.

TABLE 4 SPORE #20: MICROENCAPSULATION TAXOL PK STUDY Treatment Day Day 2Day 4 Day 6 Day 8 20 May 1997 23 May 1997 28 May 1997 01 Jun. 1997 25Jun. 1997 5 Jul. 1997 Animal # Weight (gm) Weight (gm) Weight (gm) WBCWBC Weight (gm) WBC Weight (gm) WBC Cage 1 0.04 mg/kg subcut 1 16.1518.00 18.65 5.9 2 17.97 18.10 19.68 19.77  4.1 3 18.16 19.20 17.40 17.4317.30 4.5 4 18.19 16.19 20.00 20.18 20.50 20.80 5.0 Cage 2 0.4 mg/kgsubcut 1 16.00 18.00 20.21 7.8 2 16.97 18.82 20.23 20.37 *3 3 18.0418.25 19.21 19.49 19.78 8.3 4 17.80 17.67 18.07 18.91 19.15 19.26 8.0Cage 3 2.0 mg/kg subcut 1 17.44 18.04 18.05 4.9 2 18.00 18.04 18.0118.20  6.1 3 14.80 15.26 16.25 18.38 16.93 4.3 4 17.71 17.90 20.00 20.7020.83 20.96 8.6 Cage 4 4 mg/kg subcut 1 17.60 18.10 19.87 *3.1 2 19.4720.13 21.23 21.92  2.5 3 15.78 15.80 21.11 21.50 21.70 6.1 4 16.92 16.9018.31 18.51 19.30 19.23 6.5 Cage 5 8 mg/kg subcut 1 17.09 17.12 19.025.9 2 19.70 19.92 18.14 19.22  7.2 3 17.00 17.10 19.04 19.30 19.22 4.5 416.98 17.00 17.82 19.40 18.32 18.54 *3.8 Cage 6 16 mg/kg subcut 1 17.5417.62 18.32 17.91  6.1 2 18.11 18.00 19.40 21.30 20.39 5.3 3 16.80 16.8021.09 17.01 21.08 20.81 *3.5 4 15.98 15.90 16.12 6.4Taxol Quantitation in Mouse Serum Samples Performed by USADRD-WRAIRA. Development of HPLC Procedure: Analyzing Taxol Content in House Serum

1. Procedure

-   -   a. Taxol stock (1.06 mg/ml) was prepared in 100% acetonitrile.    -   b. A set of taxol standards were made in 100% acetonitrile (0.53        ug/ml-106 ug/ml) from the taxol stock for use to generate a        taxol calibration curve.    -   c. A set of taxol standards were made in mouse serum (Sigma)        from the taxol stock (5.30 ug/ml-106 ug/ml).    -   d. Taxol/serum standards were prepared for subsequent HPLC        analysis via 2 procedures. Procedure 1 was extraction of taxol        via acetonitrile precipitation of serum proteins, and procedure        2 was extraction of taxol via solid phase extraction (SPE).    -   e. Acetonitrile Precipitation: Taxol/serum standards were        diluted 1:2 or 1:10 using acetonitrile and centrifuged for 5        minutes at 6,000 rpm. The supernatant was collected for HPLC        analysis. Sample volume for HPLC analysis was 700 ul.    -   f. Solid Phase Extraction: 5 ml of a 90:10 distilled        water/acetonitrile solution were added to SPE columns        (lgC18;6cc; Extra sep column; Thomson Instrument Company) to wet        the columns. 100 ul of taxol/serum standard (1:10 dilution) or        500 ul of taxol/serum standard (1:2 dilution) were added to the        SPE column. Serum proteins were washed from the SPE column using        90:10 distilled water/acetonitrile, and taxol was eluted from        the SPE. column using 1 ml of acetonitrile. 700 ul of        taxol/acetonitrile were used for subsequent HPLC analysis.

2. HPLC Method

-   -   a. Mobile 50—50 acetonitrile/distilled water with 0.1%        phosphoric acid        -   b. Column: PFP, 5 micron, 250×4.6 mm (Column Engineering,            Inc.)    -   c. Flow Rate: 1 ml/min; isocratic    -   d. 4 Wavelength: 227 nm        2. Results: Percent Efficiency of Taxol Recovery from        Taxol/Serum Standards

a. Acetonitrile Precipitation: 1:2 dilution

-   -   5.30 ug/ml=61.43%    -   10.6 ug/ml=55.17%    -   26.5 ug/ml=54.29%    -   53.0 ug/ml=55.18%    -   106 ug/ml=51.60%

b. Acetonitrile Precipitation: 1:10 dilution

-   -   5.30 ug/ml=121.89%    -   10.6 ug/ml=108.21%    -   26.5 ug/ml=104.98%    -   53.0 ug/ml=101.87%    -   106 ug/ml=99.27%

c. Solid Phase Extraction: 1:2 dilution

-   -   5.30 ug/ml=29.87%    -   10.6 ug/ml=29.85%    -   26.5 ug.ml=25.21%    -   53.0 ug/ml=23.67%    -   106 ug/ml=22.86%

d. Solid Phase Extraction: 1:10 dilution

-   -   5.30 ug.ml=128.49%    -   10.6 ug.ml=115.85%    -   26.5 ug/ml=63.36%    -   53.0 ug/ml=59.06%    -   106 ug/ml=3.34%

e. This experiment was repeated with similar taxol recovery rates.

3. Conclusions

a. Efficiency of taxol recovery from acetonitrile precipitation washigher than that from solid phase extraction, although a smaller SPEcolumn may also be effective.

b. Serum protein interference appeared to,be minimized using theacetonitrile precipitation (1:1O dilution) method.

c. Method selected for subsequent use was: acetonitrile precipitation(1:10 dilution).

3. Validation of Internal Standard Incorporation into the

1. Procedure

a. A 1 mg/ml stock solution of N-nitrosodiphenylamine (NDA), theinternal standard, was prepared in 100% acetonitrile.

b. A 30 ug/ml NDA solution was prepared from the NDA stock solution in100% acetonitrile.

c. A set of taxol standards in mouse serum (Sigma) were prepared in 1.5ml microfuge tubes containing NDA (100 ul of 30 ug/ml stock) and taxol.Taxol standard concentrations were as follows: 1.11 ug/ml, 2.22 ug/ml,3.33 ug/ml, 5.55 ug/ml, and 11.1 ug/ml.

d. Standards were spun for 1 min at 7,000 rpm, and 700 ul of theresulting supernatant were analyzed via HPLC.

e. 2 calibration curves were prepared, one using NDA as the internalstandard and the other using taxol alone. Calibration standardsused.were: 1.0 ug/ml, 2.0 ug/ml, 5.0 ug/ml, and 10 ug/ml. The 3.0 ug/mlstandard was run 10 times, and the amount was quantitated using eachcalibration curve.

2. HPLC Method

a. Same method as used in Section A.

3. Results

a. Calibration Curve Without NDA

-   -   1)R2=0.98533 (all standard points included)    -   2)Average concentration of the 10-3.33 ug/ml runs=2.946 (88.45%        efficiency)

b. Calibration Curve With NDA

-   -   1)R2=0.999535 (all standard points included)    -   2)Average concentration of the 10-3.33 ug/ml runs=3.246 (98.36%        efficiency)

4. Conclusions

-   -   a. Accuracy is significantly improved using the internal        standard. This internal standard will be used to analyze the        serum samples from the in vivo experiment.        C. Taxol Quantitation in Mouse Serum Samples

1. Sample Preparation

-   -   a. 100 ul of serum were transferred to a microfuge tube.    -   b. 25 ul of NDA stock (2.12 ug/ml) were added to the serum.    -   c. 876 ul of acetonitrile were added to the serum/NDA.    -   d. The microfuge tube was vortexed for 30 seconds and then        centrifuged for 30 seconds at 7,000 rpm.    -   e. 600 ul of the resulting supernatant was transferred to an        HPLC vial for analysis.    -   f. Taxol standards were prepared in serum. Total volume was        1 ml. Taxol standard concentrations were: 28 ng/ml, 56 ng/ml, 84        ng/ml, and 112 ng/ml.

note: Many of the serum samples were moderately—grossly hemolyzed.

2. BPLC Methods Applied

a. Method 1

-   -   (1) Mobile Phase: 50—50 acetonitrile/distilled water with 0.1%        phosphoric acid    -   (2) Column: PFP, 5 micron, 250×4.6 mm (Column Engineering,        Inc.).    -   (3) Flow Rate: 1 ml/min; isocratic    -   (4) Wavelength: 227 nm    -   (5) Results: In comparison with taxol standards, a peak which        appeared to be composed of 2 elements was detected at the        retention time of taxol; however, since the peak: noise ratio        was so low, identification of the peak(s) as taxol was not        possible. Additionally, due to interference from a component in        the sample, quantatation of the peak(s) was not possible.    -   (6) Additional Parameters Tested using this method:    -   Increased acetonitrile concentration up to 75% in order to        resolve the multi-element peak; however, no increase in peak        resolution was observed.

b. Method 2

-   -   (1) Mobile Phase: 70:3O acetonitrile/distilled water with 0.1%        phosphoric acid    -   (2) Column: Inertsil; 5 um; ODS-2; 250×3 mm ID    -   (3) Flow Rate: 0.4 ml.min isocratic    -   (4) Wavelength: 227 nm    -   (5) Results: This column was tried in order to increase peak        resolution and to positively identify the peak(s) in question.        We were not able to accomplish this task; however; we were able        to produce a calibration curve using mouse serum (Sigma) in the        nanogram range. R2 for this calibration curve was 0.991273.        Again, concentrations of taxol were much too low for analytical        analysis.

(c) Method 3

-   -   (1) Mobile Phase: 60:40 acetonitrile/distilled water with 0.1%        phosphoric acid    -   (2) Column: Inertsil; 5 um; ODS-2, 250×3 mm ID    -   (3) Flow Rate: 0.4 ml/min    -   (4) Wavelength: 227 nm    -   (5) Results: We were able to separate the multi-element peak        slightly; however, this separation was not yet optimal, and the        band broadening affected the integration of the taxol peak at        low nanogram levels. We were able to produce a calibration curve        using mouse serum (Sigma) in the nanogram level; however,        integration was impossible due to the band broadening effect.        Again, concentration of taxol were much too low for analytical        analysis.

d. Method 4-Gradient Method

-   -   (1) Mobile Phase: Solvent A=distilled water with 0.15 phosphoric        acid; Solvent B=acetonitrile    -   (2) Columns and Flow Rates: Intertsil ODS, 3 mm ID(0.4 ml/min);        Reliasil ODS, 3 um, 100 A, 2.1 mm ID×150 (0.4 ml/min; Reliasil        ODS 5 um, 300 A, 2.1 mm ID×150(0.2 ml/min)    -   (3) Gradient: 40-80% solvent B in 20 min    -   (4) Wavelength: 227 nm    -   (5) Results: Separation was not effective.

e. Method 5

-   -   (1) Mobile Phase: 65:35 acetonitrile/distilled water with 0.1%        phosphoric acid    -   (2) Column: Inertsil; 5 um; 58.ODS-2; 25O×3 mm ID    -   (3) Flow Rate: 0.4 ml/min; isocratic using a photodiode array        detector    -   (4) Wavelength: 227 nm    -   (5) Results: Separation of the multi-element peak was        accomplished; however, due to interference and the extremely low        taxol concentrations, positive identification/quantitation was        not possible. FIGS. 21 and 22 show representative chromatograms        of our best effort to give an indication of the range that we        had to work with. Taxol retention time was 5.572 minutes, and        NDA retention time was 8.652 minutes (FIG. 21B). Working at the        nanogram level makes background peaks more important, in terms        of their contribution to the taxol peak height and area.        Analyzing an HPLC grade acetonitrile blank showed interference        which amounted to approximately 23% of the area of the taxol        peak in the 100 ng/ml standard, while no interference with the        NDA peak was observed (compare FIG. 21A to. FIG. 21B). FIG. 22        shows chromatograms of serum samples G6#1(16 mg/ml; s.c. route;        group 1) and G12#1(16 mg/ml; i.m. route; group 1)—Taxol        concentrations were just too low to reliably work with (FIG.        22).

3. Conclusions

-   -   a. Trace amounts of taxol (in the low nanogram range) in the        serum samples were detected; however, concentrations were just        too low to reliably support this observation. A more        concentrated sample/larger sample is required to increase the        peak: noise ratio, thus, improving quantitation ability. Our        next experiment using higher concentrations of taxol may allow        us to verify that taxol is leaving the depot site and entering        the systemic circulation. Additionally, peak interference was a        problem. This interference could have been due to: (1)        acetonitrile; (2) serum protein(s);(3) specimen hemolysis; (4)        taxol metabolites. Methodologies to further “clean” up the        sample need to be employed.    -   b. Taxol is metabolized quickly; therefore, detection of parent        drug may be difficult due to interference with taxol        metabolities. Amplification of the taxol signal may be        accomplished using W chromophore derivatization specific for        functional chemical groups within the parent drug. These UV        chromophores may also amplify metabolite detection.        Additionally, serum protein(s)/acetonitrile interference with        out peak of interest in the nanogram range may be eliminated        with amplification of the desired signal.

4. Additional Steps During Sample Preparation To Overcome Problems

-   -   a. Using a smaller bed SPE column and/or liquid/liquid        extraction using diethyl ether. These steps would allow for a        smaller reconstitution volume, which may increase sensitivity.    -   b. Signal amplification using W chromophore derivatization.        BIBLIOGRAPHY

-   1. Young, J. L., Jr. 1989. Incidence and mortality of breast cancer    in Breast Cancer, B. J. Kennedy, editor. Alan R. Liss, Inc., New    York, N.Y. p.105.

-   2. Wood, W. C. 1989. Definitive surgery for stages I and II breast    cancer in Breast Cancer, B. J. Kennedy, editor, Alan R. Liss, Inc.,    New York, N.Y. p.95.101.

-   3. Boyages, J. and J. R. Harris. 1989. Conservative surgery and    radiation therapy for stages I and II breast cancer in Breast    Cancer. B. J. Kennedy, editor. Alan R. Liss, Inc., New York, N.Y.    p.102-111.

-   4. Rausch, D. J., T. Kiang, and B. J. Kennedy. 1989. Guidelines for    management of breast cancer: a treatment summary in Breast    Cancer, B. J. Kennedy, editor. Alan R. Liss, Inc., New York, N.Y.    p.225-239.

-   5. Chu, F. F. C. 1987. Radiation therapy following local excision or    partial mastectomy in Breast Cancer: Diagnosis and Treatment: I-M.    Ariel R. Liss, Inc., New York, N.Y. p.43-161.

-   6. Abrams, J. S. and J. Aisner. 1989. The evolving approach to stage    III breast cancer in Breast Cancer, B. J. Kennedy, editor. Alan R.    Liss, Inc., New York, N.Y. p.43-161.

-   7. Booser, D. J. and G. N. Hortogagy. 1992. Treatment of locally    advanced breast cancer. Semin. Oncol. 19(3): 278-285.

-   8. Fowble, B. and D. Glover. 1991. Locally advanced breast cancer in    Breast Cancer Treatment: A Comprehensive Guide to Management. B.    Fowble, R. L. Goodman, J. G. Glick, and E. F. Rosato, editors. Mosby    Year Book, St. Louis, Mo. p.345-372.

-   9. Henderson, I. C. 1989. Adjuvant systemic therapy of breast cancer    in Breast Cancer, B. J. Kennedy, editor. Alan R. Liss, Inc., New    York, N.Y. p.113-141.

-   10. Taylor, S. G., IV. 1989. Advanced breast cancer management;    chemotherapy in Breast Cancer, B. J. Kennedy, editor. Alan R. Liss,    Inc., New York, N.Y. p.173-187.

-   11. Dordunoo, S. K., J. K. Jackson, L. A. Arsenault, A. M. C.    Oktaba, W. L. Hunter, and H. M. Burt. 1995. Taxol encapsulation in    poly(e=caprolactone)microspheres. Cancer Chemother. Pharmacol.    36:279-282.

-   12. Burt, H. M., J. K. Jackson, S. K. Bains, R. T. Liggins, A. M. C.    Oktaba, A. L. Arsenault, and W. L. Hunter. 1995. Controlled delivery    of taxol from microspheres composed of a blend of ethylene-vinyl    acetate copolymer and poly(d,l-lactic acid). Cancer Lett. 88: 73-79.

-   13. Kunieda, K., T. Seki, S. Nakatani M. Wakabayashi, T. Shiro, K.    Inoue, M. Sougawa, R. Kimura, and K. Harada. 1993. Implantation    treatment method of slow release anticancer doxorubicin containing    hydroxyapatite (DOX-HAP) cinokexL a basuc study of a new treatment    for hepatic cancer. Br. J. Cancer 67:668-673.

-   14. Gasparini, A., M. Tonetti, B. Astroff, L. Rowe, W.    Satterfield, R. Schmidt, and J. R. DeLoach. 1992. Pharmacokinetics    of doxorubicin loaded and glutaraldehyde treated erythrocytes in    healthy and lymphoma bearing dogs in The Use of Resealed    Erythrocytes as Carriers and Bioreactors, M. Magnani and J. R.    DeLoach, editors. Plenum Press, New York, N.Y. p.299-304.

-   15. Kies, M. S. 1987. Adjuvant chemotherapy of breast cancer in    Breast Cancer: Diagnosis and Treatment. I. M. Ariel and J. B.    Cleary, editors. McGraw-Hill Book Company, New York, N.Y. p.328-343.

-   16. Seidman, A. D., D. Hochhauser, M. Gollub, B. Edelman, T.    Yao, C. A. Hudis, P. Francis, D. Fennely, T. A. Gilewski, M. E.    Moynahan, V. Currie, J. Baselga, W. Tong, M. O'Donahue, R.    Salvaggio, L. Auguste, D. Spriggs, and L. Norton. 1996. Ninety-six    hour paclitaxel infusion after progression during short taxane    exposure; a phase II pharmacokimetic and pharmacodynamic study in    metastatic breast cancer. J. Clin Omcol 146:1877-1884.

-   17. Sharma, A., E. Mayhew, and R. M. Straubinger. 1995. Antimor    effect of taxol-containing liposomes in a taxol-resistant murine    numor model. Cancer Res. 53:5877-5881.

-   18. Straubinger, R. M., A. Sharma, M. Murray, and E. Mayhew. 1993.    Novel taxol formulations: taxol-containing liposomes. Monogr, Natl.    Cancer Inst. .15:69-78.

-   19. Bartoli, M. H., M. Boitard, H. Fessi, H. Beriel, J. P.    Devissaguet, F. Picot. and F. Puisieux. 1990. In vitro and in vivo    tumoricidal activity of free and encapsulated taxol.    Microencapsulation 7(2):191-197.

-   20. Jampel, H. D., D. Thibault, K. W. Leong, P. Uppal, and H. A.    Quigley. 1993. Glamcoma filtration surgery in nonhuman primates;    using taxol and etoposide in polyamhydride carriers. Invest.    Ophthalmol. Vis. Sci. 34:3076-3083.

-   21. Winternitz, C. I., J. K. Jackson, A. M. Oktaba, and H. M.    Burt. 1996. Development of a polymeric surgical paste formulation    for taxol. Pharm. Res. 13(3):368-375.

-   22. Innocenti, F., R. Danesi, A. dipaolo, C. Agen, D. Nardini. G.    Bocci and M.del Tacca. 1995. Plasma and tissue disposition of    paclitaxel(taxol) after intraperitoneal administration in mice. Drug    Metabolism and Disposition 23(7): 713-717.

-   23. Lewis, D. H. 1990. Controlled release of bioactive agents from    lactide/glycolide polymers in Biodegradable Polymers as Drug    Delivery Systems. M. Chasin and R. Langer. editors. Marcel Dekker,    Inc., New York, N.Y. p1-41.

-   24. Zhifang, Z., Z.Mingxing, W. Shengao, L. Fang, and S.    Wenzhao. 1993. Preparation and evaluation in vitro and in vivo    copoly(lactic/glycolic) acid microspheres containing norethisterone.    Biomat. Art. Cells Immob. Biotech. 21(1): 71-84.

-   25. Visscher, G. E., R. L. Robinson, and G. J. Argentieri. 1987.    Tissue response to biodegradable injectable microcapsules. J.    Biomat. Applic. 2:118-131.

-   26. Toguchi, H. 1991. Formulation study of leuprorelin acetate to    improve clinical performance. Clin. Therap. 14(Suppl A):1210 128.

-   27. Balant, L. P. 1993. Regulatory aspects of modified release    dosage forms: clinical studies. Boll. Chim. Farmaceutico    1332(5):143-149.

-   28. Redding, T. W., A. V. Schally, T. R. Tice, and W. E.    Meyers. 1984. Long-acting delivery systems for peptides: inhibition    of rat prostate tumors by controlled release of (D-up) luteinizing    hormone-releasing hormone from injectable microspheres. Proc. Natl.    Acad. Sci. USA 81:5845-5848.

-   29. Moritera, T., Y. Ogura, N. Yoshimura, Y. Honda, R. Wada, S. H.    Won, and Y. Ikada. 1992. Biodegradble microspheres containing    adriamycin in the treatment of proliferative vitreoretinopathy.    Invest. Ophthalmol. Vis. Sci 33:3125-3130.

-   30. Rosen, H. B., J. Kohn, K. Leong, and Langer. 1988. Bioerodible    polymers for controlled release systems in Controlled Release    Systems: Fabrication Technology. volume II. D. Hsieh, editor. CRC    Press, Inc., Boca Raton, Fla. p.84-110.

-   31. Lewis, D. H. 1990. Controlled release of bioactive agents from    lactide/glycolide polymers in Biodegradable Polymers as Drug    Delivery Systems, M. Chasin and R. Langer, editors. Marcel Dekker,    Inc., New York, N.Y. p.1-41.

-   32. Kingston, D. G. I. 1991. The chemistry of taxol. Pharmac. Ther.    52:1-34.

-   33. Rowinsky, E. K. L. A. Cazenave, and R. C. Donehower. 1990.    Taxol: a novel investigational antimicrotubule agent. J. Natl.    Cancer Inst. 82:1247-1259.

-   34. Schiff, P. B., J. Fant, and S. B. Horwitz. 1979. Promotion of    microtubule assembly in vitro by taxol. Nature 277:665-667.

-   35. Kearns, C. M., L. Gianni, and M. J. Egorin. 1995. Paclitaxel    pharmacokinetics and pharmacodynamics. Semin. Oncol. 22(3): 16-23.

-   36. Zhifang, Z., Z. Mingxing, W. Shengao, L. Fang, and S.    Wenzhao. 1993. Preparation and evaluation of in vitro and in vivo    copoly(lactic)acid microspheres containing norethisterone. Biomet.    Art. Cells Immob. Biotech. 21(1): 71-84.

-   37. Visscher, G. E., R. L. Robinson, and G. J. Argentieri. 1987.    Tissue response to biodegradable injectable microspheres. J.Biomat.    Applic. 2:118-131.

-   38. Toguchi, H. 1992. Formulation study of leuprorelin acetate to    improve clinical performance. Clin. Therap. 14(Suppl.A): 121-128.

-   39. Balant, L. P. 1993. Regulatory aspects of modified release    dosage forms: clinical studies. Boll. Chim. Farmaceutico 132(5):    143-149.

-   40. Moritera, T., Y. Ogura, N. Yoshimura, Y. Honda, R. Wada, S. H.    Won r and Y. Ikada. 1992. Biodegradable microspheres. containing    adriamycin in the treatment of proliferative vitreoretinopathy.    Invest. Ophthalmol. Vis.Sci 33:2125-2130.

-   41. Redding, T. W., A. V. Schally, T. R. Tice, and W. E.    Meyers. 1984. Long-acting delivery systems for peptides: inhibition    of rat prostate tumors by controlled release of (D-trp6) luteinizing    hormone-releasing hormone from injectable microcapsules. proc. Natl.    Acad. Sci. USA 81:5845-5848. 66.42. Jacob, E., J. A.    Setterstrom, D. E. Black, Jr., J. R. Heath, III; and others. 1991.    Evaluation of biodegradable ampicillin anhydrate microcapsules for    local treatment of osteomyelitis in a rabbit model. Clin. Orthop.    267:237-244.

-   43. Jacob, E., G. Cierny, M. T. Fallon, J. F. McNeill, Jr.,    and G. S. Siderys. 1993. Evaluation of biodegradable cefazolin    sodium microspheres for the prevention of infection in rabbits with    experiemental open tibial fractures stabilized with internal    fixation. J. Orthop. Res. 11(3):401-411.

-   44. Setterstrom, J. A., T. R. Tice, and W. E. Myers. 1984.    Development of encapsulated antibiotics for topical administration    to wounds in Recent Advances in Drug Delivery Systems. J. M.    Anderson and S. W. Kim, editors, Plenum Publ. Corp., New York, N.Y.    p.185-198.

-   45. Rethman, M. P., J. A. Setterstrom, E. Jacob, J. R. Health,    and D. Polly. 1988. Locally applied microencapsualted ampicillin    anhydrate obvious S. aureus infection of internally fixed fractures    in rats. J. Dent. Res. (sp.Issue) 67:298.

-   46. Sharma, A., E. Mayhew, and R. M. Straubinger. 1993. Antitumor    effect of taxol-containirrg.liposomes in a taxol-resistant tumor    model. Cancer Res. 53:5877-5881.

-   47. Straubinger, R. M., A. Sharma, M. Murray, and E. Mayhew. 1993.    Novel taxol formulations: tax0l containing liposomes. Monogr. Natl.    Cancer Inst. 15:69-78.

-   48. Dordumoo, S. K., J. K. Jackson, L. A. ArSenaUlt, A. M. C.    Oktaba, W. L. Hunter, and H. M. Burt. 1995. Taxol encapsulation in    poly-(e-caprolactone) microspheres. Cancer Chemother. Pharmacol.    36:279-282.

-   49. Burt, H. M., J. K. Jackson, S. K. Bains, R. T. Liggins, A. M. C.    Oktaba, A. L. Arsenault, and W. L. Hunter. 1995. Controlled delivery    of taxol from microspheres composed of a blend of ethylene-vinyl    acetate copolymer and poly(d,l-lactic acid). Cancer Lett. 88:73-79.

-   50. Bartoli, M. H., M. Boitard, H. Ressi, H. Beriel, P.    Devissaguet, F. Picot, and F. Pusieux. 1990. In vitro and in vivo    tumoricidal activity of free and encapsulated taxol. J.    Microencapsulation 7(2): 191-197.

-   51. Kumagai, S., T. Sugiyama, T. Nishida, K. Ushijima, and M.    Yakushiji. 1996. Improvement of intraperitoneal chemotherapy for rat    ovarian cancer using cisplatin-containing microspheres. Jpn. J.    Cancer Res. 87-412-417.

-   52. Sinko, P. and J. Kohn. 1993. Polymeric drug delivery systems—an    overview ACS Symposium Series, 520:18-41.

-   53. Kunieda, K., T. Seki, S. Nakatani, M. Wakabayashi, T. Shiro, K.    Inoue, M. Sougawa, R. Kimura, and K. Harada. 1993′. Implantation    treatment and method of slow release doxorubicin containing    hydroxyapatite (DOX-HAP) complex. A basic study of a new treatment    for hepatic cancer. Br. J. Cancer 67:668-673.

-   54. Gasparini, A., M. Tonetti, B. Astroff, L. Rowe, W.    Satterfield, R. Schmidt, and J. R. DeLoach. 1992. pharmacokinetics    of doxorubicin loaded and glutaraldehyde treated erythrocytes in    healthy and lymphoma bearing dogs in The Use of Resealed    Erythrocytes as Carriers and Bioreactors. M. Magnani and J. R.    DeLoach, editors.

-   55. Henderson, I. C. 1989. Adjuvant systemic therapy of breast    cancer in Breast Cancer, B. J. Kennedy, editor. Alan R. Liss, Inc.,    New York, N.Y. p.113-141.

-   56. Kies, M. S. 1987. Adjuvant chemotherapy of breast cancer in    Breast Cancer Diagnosis and Treatment, I. M. Ariel and J. B. Cleary,    editors. McGraw-Hill Book Company, New York, N.Y. p.3280 343.

-   57. Shikan, A. H., D. W. Eisels, and A. J. Domb. 1994. Polymer    delivery of chemotherapy for squamous cell carcinoma of the head and    nect. Arch. Otolaryngol: Head Neck Surg. 120:1242-1247.

This invention relates to the design of biocompatible and biodegradablemicrospheres for novel, sustained release of hydrophobic agents alone,including paclitaxel, doxorubicin, 5-fluorouracil, camptothecin,cisplatin, metronidazole, and combinations, derivatives, or functionallyequivalents thereof, or in combination with hydrophillic agents over aperiod of up to 100 days in an aqueous physiological environment withlittle or no burst release.

Unlike currently available release systems which rely on the use offiller/additives such as gelatin, albumin, dextran, pectin, polyvinylpyrrolidone, polyethylene glycol, sugars, etc., and are still prone tolow encapsulation efficiencies and “burst effects”, this inventionachieves high encapsulation efficiency and “burst-free”, programmablesustained release is achieved. The excipients used in the formulation(PLGA) have molar compositions ranging from 100/O to 50/Olactide/glycolide with molecular weight of lo-100 kDa.

Additionally, two forms of the biocompatible, biodegradblepoly(DL/lactide-shield-glycolide) can be employed, one being the morehydrophobic end-capped polymer with the terminal residues functionalizedas esters, and the other being the more hydrophillic uncapped polymerwith the terminal residues existing as carboxylic acids.

All publications and patent applications mentioned in this specificationare indicative of the level of skill of those skilled in the art towhich this invention pertains. All publications and patent applicationsare herein incorporated by reference to the same extent as if eachindividual publication or patent application was specifically andindividually indicated to be incorporated by reference.

Although the foregoing invention has been described in some detail byway of illustration and example for purposes of clarity ofunderstanding, it will be obvious that certain changes and amodifications may be practiced within the scope of the appended claims.

1. A method of preparing a controlled release microcapsulepharmaceutical composition of burst-free sustained, programmable releaseof a hydrophobic bioactive agent over a duration of 24 hours to 100days, comprising the steps of: a) mixing a hydrophobic bioagent with ablend of end-capped and uncapped biocompatible, biodegradablepoly(lactide/glycolide) copolymer, wherein said end-capped polymer hasterminal residues functionalized as esters and said uncapped polymer hasterminal residues existing as carboxylic acids; and b) performing asolvent evaporation process to form said microcapsules.
 2. The method ofclaim 1, wherein said copolymer has a molecular weight from 10 to 100kDa.
 3. The method of claim 1, wherein said agent is selected from thegroup consisting of chemotherapeutics, antibiotics, antivirals,antiinflammatories, cytokines, immunotoxins, anti-tumor antibodies,anti-angiogenic agents, anti-edema agents, radiosensitizers, andcombinations thereof.
 4. The method of claim 1, wherein said agent isselected from the group consisting of paclitaxel, doxorubicin,5-fluorouracil, camptothecin, cisplatin, metronicdazole, andcombinations thereof.
 5. The method of claim 1, further comprisingmanipulating a ratio of said uncapped polymer and said capped polymer tomaximize core loading efficiencies of said hydrophobic bioagent.
 6. Themethod of claim 1, further comprising adjusting a percentage of saidcapped and a percentage of said uncapped copolymers to stabilize astructure of said microcapsules.
 7. The method of claim 1, furthercomprising increasing an amount of said uncapped polymer relative tosaid capped polymer to decrease a resulting size of said microcapsuleswhile incorporating said hydrophobic bioagent into said microspheres. 8.The method of claim 1, further comprising manipulating a parameter ofacceleration of release of hydrophobic bioagent by adjusting an amountof uncapped polymer, wherein by increasing said uncapped polymer, saidacceleration of release increases.
 9. The method of claim 1, furthercomprising manipulating a parameter of acceleration of release ofhydrophobic bioagent by adjusting an amount of low molecular weightpolymer wherein the greater the amount of low molecular weight polymer,the greater the acceleration of release of said hydrophobic bioagent.10. The method of claim 1, further comprising manipulating a parameterof acceleration of release of hydrophobic bioagent by adjusting externalstir rates in the mixing step, wherein by increasing external stirrates, said release rates are increased.
 11. The method of claim 10,further comprising manipulating a parameter of acceleration of releaseof hydrophobic bioagent by sonification of the polymer and hydrophobicbioagent mixture before performing the external phase stirring.
 12. Themethod of claim 1, further comprising adding sucrose to the polymer andhydrophobic bioagent mixture to increase release rates of thehydrophobic bioagent.
 13. The method of claim 1, further comprisingcooling the hydrophobic bioagent and said polymer for a first 15 minutesof said mixing to a temperature of 15° C. to form smaller microspheres.